Therapeutic agents for glomerulosclerosis

ABSTRACT

An object of the present invention is to provide pharmaceutical compositions for inhibiting mesangial cell proliferation or mesangial matrix production without causing hypercalcemia. 
     According to the present invention, therapeutic agents for glomerulosclerosis containing 1α,3β-dihydroxy-20α-(3-hydroxy-3-methylbutyloxy)-9,10-seco-5,7,10 (19)-pregnatriene as an active ingredient are provided.

This is a continuation of parent application Ser. No. 09/959,898,nationalized Nov. 13, 2001, now U.S. Pat. No. 6,506,741 of which theInternational application PCT/JP00/02958 was filed May 9, 2000, whichdesignated the United States and which international application was notpublished in English, said co-pending parent application having nowissued as U.S. patent 6,506,741.

TECHNICAL FIELD

The present invention relates to therapeutic agents forglomerulosclerosis containing1α,3β-dihydroxy-20α-(3-hydroxy-3-methylbutyloxy)-9,10-seco-5,7,10(19)-pregnatriene (hereinafter also referred to as22-oxa-1α,25-dihydroxyvitamin D₃) as an active ingredient.

BACKGROUND ART

Glomerulosclerosis is induced by irreversible progress of variousglomerular diseases of different etiologies and histopathologicalpictures. It is mainly pathologically characterized by mesangial cellproliferation or increased mesangial matrix accompanied by atrophy,degeneration or collapse of glomerular cells. Examples of the etiologiesof glomerulosclerosis include IgA nephropathy or diabetic nephropathy,which were reported to be characterized by mesangial cell proliferationor increased mesangial matrix. In order to establish a therapy forglomerulosclerosis, it seems important to clarify the mechanism of theonset or to understand the pathology. Thus, efforts have been made todevelop experimental nephritis models that experimentally induce lesionswith mesangial cell proliferation and mesangial matrix production, and arat model of anti-Thy 1 antibody-induced nephritis was prepared(Bagchus, W. M. et al., Lab. Invest, Vol. 55, No. 6, pp. 680-687, 1986).This model shows glomerular nephritis with mesangial cell proliferationand mesangial matrix production caused by the reaction between Thy 1antigen existing as a membrane protein in mesangial cells and anantibody against it, and drug efficacy tests in this model were reported(Masashi Haraguchi et al., Kidney International, Vol. 51 (1997), pp.1838-1846).

Drugs such as antitumor agents or immunosuppressive agents based onactivated vitamins D₃ (i.e., 1,25-dihydroxyvitamins D₃) have beendeveloped since they were reported to have not only a calcemic actionbut also a differentiation-inducing effect (Abe E. et al., Proc. Natl.Acad. Sci. USA, Vol. 78, No. 8, pp. 4990-4994, 1981).1,25-Dihydroxyvitamins D₃ were also reported to have anantiproliferative effect on human mesangial cells (Weinreich T. et al.,American Journal of Kidney Diseases, Vol. XVIII, No. 3, 1991, pp.359-366). However, it was difficult to use 1,25-dihydroxyvitamins D₃ forantiproliferative purposes due to hypercalcemia.

Thus, vitamin D₃ derivatives with a low calcemic action were researchedand some derivatives were developed. One of such vitamin D₃ derivativesis 22-oxa-1α,25-dihydroxyvitamin D₃, which was reported to show adifferentiation-inducing effect without causing hypercalcemia (Abe J. etal., FEBS Lett., Vol. 226, No. 1, pp. 58-62, 1987). Japanese Patent No.2854600 describes that 22-oxa-1α,25-dihydroxyvitamin D₃ inhibits urinaryprotein excretion so that it is useful as a therapeutic agent forglomerulonephritis.

However, nothing has been shown about the effect of22-oxa-1α,25-dihydroxyvitamin D₃ on mesangial cell proliferation ormesangial matrix production.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide pharmaceuticalcompositions having the effect of inhibiting mesangial cellproliferation or mesangial matrix production with a low calcemic action.

As a result of careful studies to attain the above object, the inventoraccomplished the present invention on the basis of the finding that22-oxa-1α,25-dihydroxyvitamin D₃ inhibits mesangial cell proliferationwithout causing hypercalcemia and also inhibits the expression ofcollagen in a rat model of anti-Thy 1 antibody-induced nephritis.

Accordingly, the present invention provides therapeutic agents forglomerulosclerosis containing1α,3β-dihydroxy-20α-(3-hydroxy-3-methylbutyloxy)-9,10-seco-5,7,10(19)-pregnatriene as an active ingredient.

Therapeutic agents for glomerulosclerosis of the present invention canbe used as pharmaceutical compositions for inhibiting mesangial cellproliferation or excessive mesangial matrix production.

Therapeutic agents for glomerulosclerosis of the present invention canalso be used as pharmaceutical compositions for inhibiting theoverexpression of collagen in the mesangium.

The present application claims priority based on Japanese PatentApplication No. 128566/1999, the disclosure of which is entirelyincorporated herein as reference.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 is a graph showing changes in glomerular cell count in a ratmodel treated with vitamin D derivatives. In this and the subsequentfigures, “OCT” represents a group treated with an anti-Thy 1 antibodyand 22-oxa-1α,25-dihydroxyvitamin D₃, “1α,25-dihydroxyvitamin D₃”represents a group treated with the anti-Thy 1 antibody and1α,25-dihydroxyvitamin D₃, “Disease control” represents a group treatedwith the anti-Thy 1 antibody alone, and “Normal control” represents anuntreated group.

FIG. 2 is a graph showing changes in glomerulosclerosis index in the ratmodel treated with vitamin D derivatives.

FIG. 3 is a graph showing changes in glomerular volume in the rat modeltreated with vitamin D derivatives.

FIG. 4 shows immunohistochemical results of type I collagen expressionin the control rat model.

FIG. 5 shows immunohistochemical results of type I collagen expressionin the rat model treated with the anti-Thy 1 antibody.

FIG. 6 shows immunohistochemical results of type I collagen expressionin the rat model treated with the anti-Thy 1 antibody and1α,25-dihydroxyvitamin D₃.

FIG. 7 shows immunohistochemical results of type I collagen expressionin the rat model treated with the anti-Thy 1 antibody and22-oxa-1α,25-dihydroxyvitamin D₃.

FIG. 8 shows immunohistochemical results of type IV collagen expressionin the control rat model.

FIG. 9 shows immunohistochemical results of type IV collagen expressionin the rat model treated with the anti-Thy 1 antibody.

FIG. 10 shows immunohistochemical results of type IV collagen expressionin the rat model treated with the anti-Thy 1 antibody and1α,25-dihydroxyvitamin D₃.

FIG. 11 shows immunohistochemical results of type IV collagen expressionin the rat model treated with the anti-Thy 1 antibody and22-oxa-1α,25-dihydroxyvitamin D₃.

FIG. 12 is a graph showing changes in serum calcium (Ca) in the ratmodel treated with vitamin D derivatives.

FIG. 13 is a graph showing changes in urinary albumin (uALB) in the ratmodel treated with vitamin D derivatives.

THE MOST PREFERRED EMBODIMENTS OF THE INVENTION

The present invention will now be described more in detail only forillustrative but non-limitative purposes.

1α,3β-Dihydroxy-20α-(3-hydroxy-3-methylbutyloxy)-9,10-seco-5,7,10(19)-pregnatriene (ie, 22-oxa-1α,25-dihydroxyvitamin D₃ or OCT) used asan active ingredient in the present invention is a known compound thatcan be synthesized by the process described in JPA No. 267550/86, forexample.

However, the compound that can be used as an active ingredient in thepresent invention is not limited to 22-oxa-1α,25-dihydroxyvitamin D₃,and other vitamin D₃ derivatives having the effect of inhibitingmesangial cell proliferation or mesangial matrix production with a lowcalcemic action such as 16-enevitamin D₃ derivatives can also be used.

The “mesangial cell” and “mesangial matrix” are explained below.

The mesangium is a tissue supporting capillary loops in the glomerulusof the kidney and composed of mesangial cells and mesangial matrix.Mesangial cells are known to maintain the loop structure of theglomerulus as well as have a phagocytic function or the ability toregulate glomerular blood flow. Mesangial cells have angiotensin IIreceptors and produce platelet-activating factor, prostaglandin, type IVcollagen, fibronectin, etc. The mesangial matrix is an extracellularmatrix component that surrounds mesangial cells.

The pharmacological effect of 22-oxa-1α,25-dihydroxyvitamin D₃ used asan active ingredient in the present invention can be evaluated on a ratmodel of a anti-Thy 1 antibody-induced nephritis characterized byshowing increased mesangial cell proliferation and mesangial matrixproduction after administration of an anti-Thy 1 antibody. Thepharmacological effect of test compounds can be evaluated byadministering an anti-Thy 1 antibody to such a rat model followed by thetest compounds, collecting renal tissue at an appropriate time anddetermining the glomerular cell count or glomerulosclerosis index ordetermining the expression of collagen by a standard method such as animmunohistochemical technique.

The mode of administration of pharmaceutical compositions of the presentinvention is not specifically limited, either oral or parenteral (forexample, intravenous, intramuscular, intraperitoneal, etc.).

Pharmaceutical compositions of the present invention are preferablyformulated and used in appropriate dosage forms including, but notlimited to, tablets, granules, fine granules, capsules, powders,injections, solutions, suspensions, emulsions, transdermal patches andsuppositories containing1α,3β-dihydroxy-20α-(3-hydroxy-3-methylbutyloxy)-9,10-seco-5,7,10(19)-pregnatriene as an active ingredient optionally in combination withpharmaceutically acceptable carriers, excipients, disintegrators,lubricants, binders, perfumes, colorants, etc.

The dose can be appropriately chosen to be effective for inhibitingexcessive mesangial cell proliferation or excessive mesangial matrix(e.g., collagen) production in the subject depending on the disease tobe treated, the condition, body type, constitution, age and sex of thepatient, and the administration route, dosage form or other factors. Thedose expressed in the weight of an active ingredient is typically in therange of 0.001-1000 μg/kg/day, preferably 0.01-100 μg/kg/day, morepreferably 0.1-10 μg/kg/day.

The following example further illustrates the present invention without,however, limiting the same thereto.

EXAMPLE 1 Test Method

A rat model of anti-Thy 1 antibody-induced nephritis was prepared, whichshowed increased mesangial cell proliferation and mesangial matrixproduction on days 4-7 after antibody administration. An anti-Thy 1antibody (OX-7 purchased from CEDARLANE, Ontario, Canada) wasadministered into the tail vein of 6-week-old male Wistar rats at 1mg/kg to induce anti-Thy 1 nephritis. Next day after OX-7administration, 22-oxa-1α,25-dihydroxyvitamin D₃ was intravenouslyadministered at 0.4 μg/kg every two days, and on day 7 afteradministration, serum, urine and renal tissue were collected.

For comparison, 1α,25-dihydroxyvitamin D₃ was administered in the samemanner instead of 22-oxa-1α,25-dihydroxyvitamin D₃. Rats not treatedwith the anti-Thy 1 antibody were used as normal controls. Rats treatedwith the anti-Thy 1 antibody alone without any vitamin D derivative wereused as disease controls.

In the renal tissue, the glomerular cell count, glomerulosclerosis indexand glomerular volume were determined (FIGS. 1, 2 and 3). The“glomerular cell count” was determined by counting every cell in everymeasurable glomerulus on hematoxylin-eosin stained samples. The“glomerulosclerosis index” was evaluated on PAM (periodicacid-methenamine-silver) stained samples according to the method of Rajiet al. The “glomerular volume” was determined by using NIH image tocalculate the volume of glomeruli read by an image analyzer.

In the renal tissue, the expression of type I collagen (ColI) and typeIV collagen (ColIV) in the mesangium was immunohistologically examined(FIGS. 4-11). Staining was performed on 4 μm sections using an ABC Eliteperoxidase staining kit (Vector Lab., Burlingame, Calif., U.S.A.)according to the manufacturer's instructions. The primary antibodiesused were a guinea pig anti-rat type I collagen antibody (BoehringerIngelheim, Co., Ltd., Ingelheim, Germany) and a rabbit anti-rat type IVcollagen antibody (Boehringer Ingelheim, Co., Ltd., Ingelheim, Germany).

In addition, serum calcium (Ca) was determined with an autoanalyzer(AUTOANALYZER, HITACHI 7170, Tokyo, Japan) and urinary albumin (uALB)was determined with an ELISA kit NEPHRAT II (EXOCELL, INC) (FIGS. 12 and13).

Results

(1) Glomerular Cell Count, Glomerulosclerosis Index and GlomerularVolume

Administration of 1α,25-dihydroxyvitamin D₃ and22-oxa-1α,25-dihydroxyvitamin D₃ significantly suppressed glomerularcell count (FIG. 1) and glomerulosclerosis index (FIG. 2) but showed noeffect on glomerular volume (FIG. 3).

(2) Immunohistochemical Analysis (ColI, ColIV)

FIGS. 4-8 and 9-11 show that administration of 1α,25-dihydroxyvitamin D3and 22-oxa-1α,25-dihydroxyvitamin D₃ clearly inhibited the expression ofColI and ColIV.

(3) Ca and uALB Levels

Administration of 22-oxa-1α,25-dihydroxyvitamin D₃ significantlysuppressed uALB levels without increasing serum Ca levels. However,administration of 1α,25-dihydroxyvitamin D₃ showed a significantincrease in serum Ca levels though uALB levels were significantlysuppressed (FIGS. 12 and 13).

INDUSTRIAL APPLICABILITY

As described above, it was shown that 22-oxa-1α,25-dihydroxyvitamin D₃inhibits mesangial cell proliferation and excessive mesangial matrixproduction in a rat model of anti-Thy 1 nephritis without increasingserum calcium, thereby inhibiting the progress of glomerulosclerosis.Therefore, the present compound is useful as a therapeutic agent forinhibiting the progress of glomerulosclerosis without inducinghypercalcemia.

1. A method of treating diabetic nephropathy, comprising administeringto a host in need of such a treatment a therapeutically effective amountof1α,3β-dihydroxy-20α-(3-hydroxy-3-methylbutyloxy)-9,10-seco-5,7,10(19)-pregnatriene.2. The method of claim 1, wherein said host is a patient in need ofinhibition of mesangial cell proliferation or mesangial matrixproduction.
 3. The method of claim 1, in which said host is a patient inneed of inhibition of expression of collagen in the mesangium.